1. Technical Field
The present invention relates to a method of preventing and reversing excitatory amino acid (EAA) initiated excitotoxicity in the central nervous system and the clinical uses thereof.
2. Description of the Prior Art
The central nervous system (CNS) is comprised of the spinal cord, brain and retina, and contains trillions of nerve cells (neurons) that form networks capable of perforning extremely complex functions. The key to the operation of these complex networks is the ability of neurons to communicate with each other. One of the ways neurons communicate with one another is by using neurotransmitters. Neurotransmitters are typically excitatory or inhibitory in nature. One class of the excitatory neurotransmitters is of particular interest.
Excitatory amino acids (EAAs) mediate a substantial portion of the chemical synaptic activity occurring in the CNS. However, several EAAs, like glutamate for example, which function under normal and healthy conditions as an important excitatory neurotransmitter in the CNS, can exert neurotoxic properties referred to as "excitotoxicity" if certain conditions arise.
It has long been recognized that excitatory amino acids may be neurotoxic Lucas and Newhouse, Arch. Ophtalmol., 58:193 (1957); Olney et al, Exp. Brain Res., 14:61 (1971); Coyle et al., Neurosci. Res. Prog. Bull., 19:331 (1981)!. Prolonged exposure of cerebral neurons to high concentrations of L-glutamate or related amino acids leads to their death and degeneration D. W. Choi, Neuron, 1:623-634, (1988); J. W. Olney, Science, 164:719-721, (1969)!. In early studies of this phenomenon, Olney showed that the neurotoxic properties of acidic amino acids were related to their ability to depolarize and excite central neurons, and the term "excitotoxicity" was coined to describe this form of activity-dependent neuronal damage. It has been suggested that these so-called "excitotoxins" may contribute to tissue damage within the central nervous system in a variety of disorders including epilepsy, neurodegenerative diseases and cerebral ischemia B. Meldrum, Clin. Sci., 68:113 (1985); M. Pisa et al., Brain Res., 200(2):481-7 (1980)!.
Current understanding recognizes at least three major ionotropic receptors for EAAs. Most commonly identified by prototypical agonists, these include: 1) receptors activated by AMPA, a cyclic analog of L-glutamate (GLU), 2) receptors activated by the neurotoxin kainic acid (KA), and 3) receptors responding to N-methyl-D-aspartate (NMDA), a synthetic analog of L-aspartate D. R. Curtus at el., Brain Res., 41, 283-301 (1972); J. C. Watkins and R. H. Evans, Ann. Rev. Pharmacol. Toxicol., 21, 165-204 (1981); A. C. Foster and G. Fragg, Brain Res. Rev., 7, 103-164 (1984)!. In addition, evidence now suggests the presence of metabotropic EAA receptors which directly activate second messenger systems D. Schoepp, J. Brockart and F. Sladeczek, In C. Lodge and G. L. Collinridge (eds.) Tr. Pharmacol. Sci., Special Report, "The Pharmacology of Excitatory Amino Acids," Elsevier, Cambridge, UK, p 74-81 (1991)!. Furthermore, it is also now apparent that the NMDA-mediated ionotropic receptors are subject to complex regulatory influences and, that this particular recognition site may exist as a supramolecular entity similar to the GABA/benzodiazepine/barbiturate effector proteins E. Costa, Neuropsychopharmacology, 2, 167-174 (1989)!.
In general, EAA agonists are potent convulsants. Additionally, AMPA, KA and the endogenous NMDA agonist, quinolinic acid (QA) and the mixed ionotropic/metabotropic agonist ibotenic acid have been used to produce laboratory models of neurodegenerative disorders K. Biziere et al. In H. Yoshida, Y. Hagihara and S. Ebashi (eds.), "Advances in Pharmacology and Therapeutics," New York: Pergamon, 1982, 271-276; R. Schwarcz, E. O. Whetsell and R. M. Mango, Science, 219, 316-318 (1983)!. As mentioned above, it has also been suggested that dysfunctions in EAA neurotransmission may contribute to the neuropathologies associated with the epilepsies and neurodegenerative conditions B. Meldrum and M. Willams (eds.), "Current and Future Trends in Anticonvulsant, Anxiety and Stroke Therapy," New York: Wiley Liss, 1990!.
Dysfunction between the excitatory and inhibitory amino acids have also been implicated in the induction of the neurodegenerative process observed in motor neurons diseases (MND) such as in amyotrophic lateral sclerosis (ALS) V. O. Gardner et al., Spine, 15:858-863 (1990)!. ALS is characterized clinically by progressive weakness and wasting of muscles caused by a slow loss of large and small neurons, predominantly motor neurons, in the ventral spinal cord, brainstem, and motor cortex. Previous studies have established that this disease is associated with increased glutamate concentrations in the CNS as well as a loss of high-affinity glutamate transport in certain brain regions and the spinal cord of affected patients J. D. Rothstein et al., Ann. Neurol., 28:18-25 (1990); A. Plaitakis et al., Ann. Neurol., 24:446-449 (1988); J. D. Rothstein et al., N. Engl. J. Med., 236:1464-1468 (1992)!. These results suggest that the defect in glutamate transport is responsible for the sustained elevations in extracellular glutamate, which in turn would result in the injury of nearby neurons. In support for this, in ALS patients, NMDA receptor binding is reduced in the ventral horn (and also the dorsal horn) of the spinal cord, presumably due to receptor down-regulation or excitotoxic degeneration of receptor-bearing cells C. Krieger et al., Neurosci Lett., 159:191-194 (1993); P. J. Shaw et al., Brain Res., 637:297-302 (1994)!.
The development of selective EAA antagonists has further expanded the understanding of EAA neurotransmission and the resulting physiology and pathophysiology in the mammalian brain. In particular, it has been shown that NNIDA receptor antagonists possess neuroprotective properties and can alter the course of excitotoxic degeneration I. Massieu et al., Neuroscience, 55:883-892 (1993)!. Substantial preclinical evidence is now available suggesting that NMDA receptor antagonists may be useful as anxiolytics, anticonvulsants, antiemetics European Patent Application No. 432,994!, antipsychotics or muscle relaxants, and that these compounds may prevent or reduce neuronal damage in instances of cerebral ischemia, hypoxia, hypoglycemia or trauma R. P. Simon et al., Science, 226:850-852 (1984); D. N. Stephens et al., Psycopharmacology, 90:166-169 (1986); D. Lodge and G. L. Collingridge (eds.) "The Pharmacology of Excitatory Amino Acids," Elsevier Trends Journals, Cambridge, UK. (1991); A. J. Faden et al., Eur. J. Pharmacol., 175:165-174 (1990)!. Likewise, it has been shown that motor neuron toxicity was selectively prevented by non-NMDA glutamate receptor antagonists in models of ALS and other MND which are associated with slow neurotoxicity J. D. Rothstein et al., Proc. Natl. Acad. Sci., 90:6591-6595 (1993)!. Excitatory amino acid antagonists have also been shown to have an analgesic effect. An initial report by Cahusac et al. indicated that intrathecal (i.t.) injection of APV, a selective NMDA antagonist, produced antinociceptive effects in tail-flick, hot-plate and paw pressure tests P. M. B. Cahusac, R. H. Hill et al, Neuropharmacology, 23:719-724 (1984)!.
Unfortunately, the levels of EAA antagonists necessary to produce the desired effects can be quite high. For example, Cahusac et al. observed that i.t. doses between 12 and 48 .mu.g induced motor dysfunction including paralysis, while doses as high as 500 .mu.g have been used in studies of the antinociceptive effects of APV in persistent nociceptive models P. M. B. Cahusac, R. H. Hill et al, Neuropharmacology, 23:719-724 (1984); T. J. Coderre and I. Van Empel, Pain, 59:345-352 (1994)!. Furthermore, competitive NMDA antagonists such as APV do not cross the blood-brain barrier so clinical administration is limited, and non-competitive NMDA antagonists such as ketamine, PCP and MK-801 produce psycomotor effects at the higher dose levels and may have a limited effective dose range.
The adrenal medulla is composed of chromaffin cells supported by connective tissue elements and profusely supplied by nerves and blood vessels. Ganglion cells are present but are usually difficult to find in routine sections. Chromaffin cells are derived from neuroectoderm and were generally thought to release catecholainines (epinephrine and norepinephrine). In recent years, however, it has been discovered that chromaffin cells release many different neuroactive substances. Using antisera against chromogranins A, B, and C and neuropeptide Y, it was demonstrated that these antigens are costored with chromaffin vesicles. Steiner, Schimid, Fisher-Colbrie et al., Histochemistry, 91:473-477 (1989)!. In a series of papers, Boinmer and Herz reported that Met!- and Leu!enkephalin are released together with catecholamines from cultured bovine adrenal chromaffin cells M. Bommer and A. Herz, Life Sci., 44:327-335, (1989); M. Bommer and A. Herz, Neuropeptides, 13:243-251 (1989)!. Furthermore, chromaffin cells have been shown to release a variety of trophic factors as well. In fact, Unsicker described the release of substances from chroinaffin cells as a "trophic cocktail" Unsicker et al., Exp. Neuro., 123:167-173 (1993)!. As a result, adrenal medullary cells have been used in attempts to treat Parkinson's disease, Chronic Pain, and to stimulate and promote the survival of other peripheral and CNS neurons.
However, adrenal chromaffin cells are not the only type of tissue to be used in neural transplantation. Both fetal and adult striatal, cortical and tectal cells have been used as well. Furthermore, reports of cross-species (xenograft) implants are also common place in the technical literature. Also, genetically altered cells, such as nerve growth factor (NGF) producing fibroblasts, have been used as a source of graft material.
Much of the literature in the field of neurotransplantation pertains to the ability of grafted material to survive in the host. With allografts, the problem of cell survival can usually be dealt with using an immunosuppressant, such as Cyclosporin A. However, attempts at cross-species transplantation have shown that such simple solutions are not adequate. Many different methods of promoting cross-species graft survivability have been demonstrated. One such method involved surrounding the graft in a polymer capsule P. Aebischer et al., Exp. Neurol., 126:151-8 (1994); J. M. Joseph et al., Cell Transplant., 3(5):355-64 (1994)!. Others involve supplying the graft with growth factors by co-grafting the implant with another cell type which releases the `necessary` growth factors in vivo G. Bing et al, Brain Res. Bull., 20:399-406 (1988); J. Kordower et al., J. Neurosurg., 73:418-428 (1990)!. Finally, it has also been established that certain cell types can survive in the CNS following short-term courses of immunosuppression if isolated from their native support cells (ie., fibroblasts and endothelial cells) J. Ortega et al., Cell Transplant., 1:33-41 (1992); S. Schueler et al., Cell Transplant., 4(1):55-64 (1995)!.
Perhaps the most fundamental question which remains concerns the mechanisms of action which underlie behavioral and morphological recovery. One of the most comprehensive perspectives is in a review by Sanberg et al. P. R. Sanberg et al., Cell Transplantation, 1(6):401-427 (1992)!. They described five alternative mechanisms of graft function which are characterized by increasingly more complex forms of host-graft interaction.
1) Nonspecific or negative consequences of the transplantation procedure. 2) Trophic actions on the host brain. Transplanted tissue may serve as a trophic support structure which exerts its behavioral protection by promoting regeneration or minimizing the extent of secondary cell loss. For example, Sanberg et al. described the use of polymer encapsulated bovine chromaffin cells (BAC) to prevent quinolinic acid induced lesions of the striatum. In their experiment, they implanted chromaffin cells into the striatum of rats as a prophylactic measure. They then tried to induce neurodegeneration by injecting quinolinic acid (QA), an endogenous EAA agonist, into the striatum. The results ofthis experiment was that the animals which received BAC implants showed little neuronal loss after the injection of QA, presumably due to the release of trophic factors P. R. Sanberg et al., Soc. Neurosci. Abstr., 17:903 (1991)!. As another example, Frim et al. conducted an experiment using a genetically altered fibroblast cell line. Here, they compared that ability of NGF (nerve growth factor, a trophic factor) producing fibroblasts vs. control fibroblasts to protect against subsequent injection of an NMDA-receptor agonist. The results again were that the NGF producing fibroblasts prevented the damage caused by the excitotoxin D. M. Frim et al., Neuroreport, 4(6):655-8 (1993)!. 3) Diffuse release of hormones or nuerotransmitters. A generalized release of nonspecific neurotransmitters or hormones into the damaged region could likely play a role in mediating recovery in several models. For example, Winnie, Sagen and Pappas have been using the fact that adrenal medullary tissue releases catecholamines and met-enkephalin to treat pain in cancer patients Winnie et al., Anesthesiology, 79(4):644-53 (1993)!. Likewise, Aebischer and co-workers have been using dopamine-secreting PC12 cells in promoting recovery in models of Parkinson's Disease P. Aebischer et al., Exp. Neurol. 111 :269-275 (1991); P. Aebischer et al., Biomaterials, 12:50-56 (1991)!. 4) Reinnervation of host tissue by the transplant. Reinnervation of host tissue has been suggested to underlie recovery in a variety of model systems by providing a tonic, unregulated release of neurotransmitters. Indeed, grafted tissue is capable of innervating large regions of the host brain, and in some cases results in a pattern of innervention which is remarkably similar to that normally observed. For example, Sanberg et al. examined the use of fetal striatal tissue to reverse the effects of a excitotoxin induced lesions as a model for Huntington's disease. In a series of experiments they concluded that the transplants not only had similar pharmacological properties but also a continuing functional interaction between the host brain and transplanted tissue which was a vital element in the success of the fetal striatal transplants A. B. Norman et al., Neuropharmacology, 27(3):333-6 (1988); P. R. Sanberg et al., J. Neural Transplant., 1(1):23-31 (1989)!. 5) Establishment of reciprocal graft-host anatomical connections. Complex behaviors with requirements for both afferent and efferent circuitry would not be modified by grafted tissue which reinnervated the host tissue but did not receive reciprocal connections itself. In general, the innervation of grafted tissue by host afferents is poor and not observed very often.
However, the use of chromaffin cells or any other living cells in a method of providing EAA antagonists into the CNS has not heretofore been attempted. Nor has the use of living cells or tissue which secrete EAA antagonists to treat dysfunctions in EAA neurotransmission heretofore been attempted. Furthermore, the use of transplants which act as EAA antagonists to reverse neurodegeneration caused by EAAs has also not been previously attempted. Finally, the ability of living cells to secrete EAA antagonists in quantities sufficient to produce neuroprotective and neuroregenerative effects without also causing locomotor abnormalities yields new and surprising results not suggested in the prior art.